Candida albicans is one of the most significant fungal/yeast pathogens in man. The organism is a normal commensal in humans, found in the gastrointestinal tract and vagina. In immunocompetent individuals Candida can colonize the oral mucosal surface (thrush) and the vaginal epithelia (Candida vaginitis). Lymphocytes are important in protection against these superficial infections consistent with the fact that HIV infected individuals are highly susceptible to mucocutaneous candidiasis.
To do more than colonize the epithelial surface, Candida need an immunocompromised host. Candidiasis is the most frequently encountered systemic fungal infection in the compromised host. For example, disseminated candidiasis occurs in 11-27% of patients with protracted neutropenia from acute leukemia or bone marrow transplantation, resulting in mortality which may approach 95%. Human immunodeficiency virus-infected patients commonly get chronic, superficial Candida infections. Increasingly,
Candida infections are encountered in seriously ill hospitalized patients without classic immunocompromising diseases. For reviews, see Greenfield, J. of Med. and Vet. Mycology 30:89-104 (1992); and Odds, ASM News 60 (6 ):313 (1994).
There is little humoral component in the immune response to systemic C. albicans infection, and protection is thought to be largely at the level of inflammation, with recruitment of neutrophils, monocytes and eosinophils to the site of infection.
Current therapy choices for fungal pathogens are limited largely to azole compounds such as clotrimazole or fluconazole, and in the case of deep systemic infections, to the highly toxic amphotericin B and 5-fluorocytosine. In addition, diagnosis of systemic candidiasis is difficult, with blood cultures often negative in spite of continued deep organ infection.
Acute inflammatory reactions are characterized by a series of vascular events including neutrophil adherence to endothelium, neutrophil diapedesis, and vasoconstriction accompanied by increased vascular permeability. Leukotrienes are important lipid derived mediators of inflammation in humans, frequently acting as powerful proinflammatory agents. Of the leukotrienes, Leukotriene B.sub.4 (LTB.sub.4, (5S, 12R)-5,12-dihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid) is a major mediator of leukocyte activation. Its effects include stimulation of cell aggregation, lysosomal enzyme release, and nondirected migration and chemotaxis. It is responsible for local recruitment of neutrophils to sites of inflammation, and is thought to be important in modulation of T lymphocyte function.
LTB.sub.4 is particularly potent in inducing leukocyte aggregation; it produces responses at concentrations in the nanomolar or high picomolar range.
The leukotrienes are synthesized by the action of lipoxygenase enzymes on arachidonic acid, which is a C.sub.20 fatty acid with double bonds at the 5-6, 8-9, 11-12, and 14-15 positions. Thus arachidonic acid is considered the precursor of all the leukotrienes.
Specifically, it appears that LTB.sub.4 is made by the conversion of LTA.sub.4, an unstable intermediate, to LTB4 by LTA.sub.4 hydrolase. LTA.sub.4 hydrolase has been cloned from humans (Funk et al., Proc. Natl. Acad. Sci. USA 84(19):6677-81 (1987)); rats (Mkita et al., FEBS Lett. 299(3):273-7 (1992); guinea pigs (unpublished; Genbank) and mice (Medina et al., Biochem. biophys. Res. Comm. 176(3):1516-24 (1991)). The mouse LTA.sub.4 hydrolase enzyme has been shown to be a zinc metalloenzyme with dual enzymatic activities, a LTA.sub.4 hydrolase activity and an aminopeptidase activity (Wetterholm et al., Biochim. et Biophys. Acta 1080(2):96-102 (1991); Orning et al., J. Biol. Chem. 269(15):11269-73 (1994)). The human LTA.sub.4 hydrolase enzyme has been shown to work both intracellularly and transcellularly to convert LTA.sub.4 to LTB.sub.4 (McGee et al., Proc. Natl. Acad. Sci. USA 83:1349-1353 ( 1986)).
It is an object of the present invention to provide for recombinant yeast LTA.sub.4 hydrolase proteins and variants thereof, and methods to produce useful quantities of these yeast LTA.sub.4 hydrolase proteins using recombinant DNA techniques.
It is a further object of the invention to provide recombinant nucleic acids encoding yeast LTA.sub.4 hydrolase proteins, and expression vectors and host cells containing the nucleic acid encoding the yeast LTA.sub.4 hydrolase protein.
An additional object of the invention is to provide monoclonal antibodies for the diagnosis of Candida infection.
A further object of the invention is to provide methods for producing the yeast LTA.sub.4 hydrolase proteins, and vaccines comprising the yeast LTA.sub.4 hydrolase proteins of the present invention.
Another object of the invention is to provide methods and compositions for screening for LTA.sub.4 hydrolase inhibitors.